Compositions and Methods for Treating Diabetes, Hypertension and Hypercholesterolemia

ABSTRACT

The present invention provides a composition comprising a therapeutically effective amount of a polypeptide, peptide, or analog corresponding to one or more of SEQ ID NO(s) 1, 2, 3 or 4, and or more of a pharmaceutically acceptable carrier, pharmaceutically acceptable diluent, and/or pharmaceutically acceptable excipient. The polypeptide therapeutic of the present invention may be formulated for administration to a subject in need of treatment as an oral formulation, a parenteral formulation, a topical formulation, an aqueous formulation, a solid formulation, a lyophilized formulation, or a trans-dermal formulation. Also disclosed is a method for treating at least one of diabetes, and/or hyperglycemia, and/or hypercholesterolemia, and/or hypertension in a subject, comprising: administering to the subject a polypeptide of the invention or formulation of the invention.

BACKGROUND OF THE INVENTION

When left uncontrolled, diabetes mellitus, or simply diabetes, resultsin hyperglycemia, or high blood sugar. Over time, hyperglycemia leads toserious damage to many of the body's systems, especially the nerves andblood vessels. There are multiple variants of diabetes. In Type 1diabetes, the pancreas produces little to no insulin. Treatment for Type1 diabetics requires insulin injections. In the most common type ofdiabetes, Type 2 diabetes, the pancreas does not produce a sufficientamount of insulin, or the insulin produced is less effective due tocellular resistance, or both. The World Health Organization providesthat Type 2 represents 90% of the cases of diabetes worldwide. Treatmentfor Type 2 diabetes includes healthy eating and physical activity, aswell as medications and insulin therapy.

Complications of the chronic hyperglycemia of diabetes includeendothelial damage, proliferative retinopathy, neuropathy, nephropathy,hypertension and ischemic heart disease. Diabetes is one of the leadingcauses of heart disease, stroke, kidney failure, blindness, and limbamputations and as such it is a drain on the economies of all industrialcountries.

Often in cases of Type 2 diabetes, pharmacological intervention isnecessary for treatment. There are many types of approved medicationsfor Type 2 diabetes, such as sulfonylureas, dipeptidyl peptidase IV(DPP-IV) inhibitors, meglitinides, biguanides, thiazolidinediones, andalpha-glucosidase inhibitors. However, these drugs produce unwanted sideeffects, including upset stomach, hypoglycemia, weight gain, liverproblems, skin rash, headache, and respiratory infection. Further, thesemedications are often used together as a combination therapy in order tobe more effective. However, the use of multiple pharmaceuticalsincreases the likelihood of unwanted side effects. Nearly 50% of Type 2diabetic patients eventually require administration of insulin.

Insulin administration remains to be the only treatment option for Type1 diabetes. Furthermore, the treatment of Type I diabetes by insulincannot avoid the long-term complications induced by daily cycles ofhyperglycemia and hypoglycemia, due to the difficulty of determining theexact insulin dosage required in changing physiological conditions.

Hypertension, high cholesterol and hyperglycemia are often present inindividuals with both types of diabetes, particularly Type 2 diabetes.The combination of hypertension, high cholesterol and diabetessignificantly increases the risk of a heart attack or stroke. Presently,the drugs used to treat these three conditions, (hypertension, highcholesterol and diabetes), have side effects to varying degrees. Itwould therefore be useful to have a therapeutic composition which lowersblood glucose also modulates blood pressure and cholesterol, withoutside effects seen in the presently available drugs. Such a compositionwould have utility in the treatment of hypertension, cholesterol, alongwith diabetes.

SUMMARY OF THE INVENTION

The present invention relates to novel pharmaceutical compositions andmethods for treating clinical diseases, including those associated withhigh blood pressure and elevated blood glucose level (hyperglycemia),such as: diabetes, stroke, peripheral vascular disease, pulmonaryhypertension, metabolic syndrome, hypercholesterolemia andatherosclerosis.

Unexpectedly, it has been discovered that a polypeptide corresponding insequence homology to an active region of a 40 s ribosomal protein S2,(“RPS2”) was beneficial as a therapeutic for oral and iv administration,as evidenced by its ability to facilitate a reduction in blood glucoselevels, a reduction in insulin resistance, a reduction in hepaticglucose production, a reduction in glucagon levels, a reduction in bloodpressure (systolic and diastolic levels), and a reduction in bloodcholesterol levels.

Provided herein are methods and compositions involving pharmaceuticalcompositions comprising RPS2 polypeptide, RPS2 peptide analogues, and/ormixtures thereof. In one embodiment, a RPS2 polypeptide comprises anamino acid sequence set forth in SEQ ID NO: 1. In another embodiment,the RPS2 polypeptide comprises an amino acid sequence having at least50% sequence identity to the amino acid sequence set forth in SEQ IDNO: 1. In yet another embodiment, the RPS2 polypeptide has at least 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequenceidentity to the amino acid sequence set forth in SEQ ID NO: 1.

In one embodiment, a RPS2 polypeptide comprises an amino acid sequenceset forth in SEQ ID NO: 2. In another embodiment, the RPS2 polypeptidecomprises an amino acid sequence having at least 50% sequence identityto the amino acid sequence set forth in SEQ ID NO: 2. In yet anotherembodiment, the RPS2 polypeptide has at least 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to the amino acidsequence set forth in SEQ ID NO: 2.

In one embodiment, a RPS2 polypeptide comprises an amino acid sequenceset forth in SEQ ID NO: 3. In another embodiment, the RPS2 polypeptidecomprises an amino acid sequence having at least 50% sequence identityto the amino acid sequence set forth in SEQ ID NO: 3. In yet anotherembodiment, the RPS2 polypeptide has at least 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to the amino acidsequence set forth in SEQ ID NO: 3.

In a first aspect, the present invention provides an isolatedpolypeptide that comprises a 40 s ribosomal protein S2 (RPS2) or afragment thereof or an analog thereto comprising an amino acid sequencehaving at least 50% sequence identity to the amino acid sequence setforth in SEQ ID NO: 1, 2, 3 or 4. In one embodiment, the presentinvention provides an isolated polypeptide that consists of a 40 sribosomal protein S2 (RPS2) or a fragment thereof comprising an aminoacid sequence having at least 50% sequence identity to the amino acidsequence set forth in SEQ ID NO: 1, 2, 3 or 4.

In one embodiment, the present invention provides an isolatedpolypeptide that comprises or consists of a 40 s ribosomal protein S2(RPS2) comprising an amino acid sequence having at least 50% sequenceidentity to the amino acid sequence set forth in SEQ ID NO: 1. In oneembodiment, the present invention provides an isolated polypeptide thatcomprises or consists of a 40 s ribosomal protein S2 (RPS2) fragmentcomprising an amino acid sequence having at least 50% sequence identityto the amino acid sequence set forth in SEQ ID NO: 2. In one embodiment,the present invention provides an isolated polypeptide that comprises orconsists of a 40 s ribosomal protein S2 (RPS2) fragment comprising anamino acid sequence having at least 50% sequence identity to the aminoacid sequence set forth in SEQ ID NO: 3. In one embodiment, the presentinvention provides an isolated polypeptide that comprises or consists ofa 40 s ribosomal protein S2 (RPS2) fragment comprising an amino acidsequence having at least 50% sequence identity to the amino acidsequence set forth in SEQ ID NO: 4.

In one embodiment, the RPS2 or fragment thereof has at least 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to theamino acid sequence set forth in SEQ ID NO: 1, 2, 3 or 4. In oneembodiment, the RPS2 has at least 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, 98% or 99% sequence identity to the amino acid sequence setforth in SEQ ID NO: 1. In one embodiment, the RPS2 fragment has at least55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequenceidentity to the amino acid sequence set forth in SEQ ID NO: 2. In oneembodiment, the RPS2 fragment has at least 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 98% or 99% sequence identity to the amino acid sequenceset forth in SEQ ID NO: 3. In one embodiment, the RPS2 fragment has atleast 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequenceidentity to the amino acid sequence set forth in SEQ ID NO: 4.

In one embodiment, the RPS2 or fragment thereof comprises the amino acidsequence set forth in SEQ ID NO: 1, 2, 3 or 4. In one embodiment, theRPS2 comprises the amino acid sequence set forth in SEQ ID NO: 1. In oneembodiment, the RPS2 fragment comprises the amino acid sequence setforth in SEQ ID NO: 2. In one embodiment, the RPS2 fragment comprisesthe amino acid sequence set forth in SEQ ID NO: 3. In one embodiment,the RPS2 fragment comprises the amino acid sequence set forth in SEQ IDNO: 4.

In one embodiment, the RPS2 or fragment thereof consists of the aminoacid sequence set forth in SEQ ID NO: 1, 2, 3 or 4. In one embodiment,the RPS2 consists of the amino acid sequence set forth in SEQ ID NO: 1.In one embodiment, the RPS2 fragment consists of the amino acid sequenceset forth in SEQ ID NO: 2. In one embodiment, the RPS2 fragment consistsof the amino acid sequence set forth in SEQ ID NO: 3. In one embodiment,the RPS2 fragment consists of the amino acid sequence set forth in SEQID NO: 4.

In a second aspect, the invention provides a formulation comprising atleast one polypeptide according to the invention. The at least onepolypeptide according to the invention may be at least one polypeptideaccording to the first aspect.

In an embodiment, the formulation is an oral pharmaceutical formulation.In an embodiment, the formulation is a pharmaceutical parenteralformulation. In an embodiment, the formulation is a pharmaceuticaltopical formulation.

In an embodiment, the formulation comprises one or more pharmaceuticallyacceptable carrier(s) and/or one or more pharmaceutically acceptablediluent(s) and/or one or more pharmaceutically acceptable excipient(s).The formulation may comprise one or more pharmaceutically acceptablecarrier(s). The formulation may comprise one or more pharmaceuticallyacceptable diluent(s). The formulation may comprise one or morepharmaceutically acceptable excipient(s).

In an embodiment, the formulation is an aqueous pharmaceuticalformulation. The peptide may be present at a concentration of 0.05 to 5μg/L in the aqueous pharmaceutical formulation. For example, the peptidemay be present at a concentration of 0.08 to 3 μg/L or may be present at0.1 to 1 μg/L. The peptide may be present at a concentration of at least0.05 μg/L, for example at least 0.7, 0.1 or 0.15 μg/L. The peptide maybe present at a concentration of not more than 5 μg/L, for example notmore than 4, 3 or 2 μg/L (e.g. not more than 1 μg/L). The aqueouspharmaceutical formulation may comprise a buffer. The buffer may have apH of from about 7 to about 8, for example the buffer may have a pH offrom about 7.2 to about 7.6, e.g. the buffer may have a physiological pH(a pH of about 7.4, e.g. a pH of from 7.3 to 7.5). The buffer may bephosphate buffered saline.

In a third aspect, the invention provides a medicament comprising apolypeptide of the invention or a formulation of the invention. Thepolypeptide of the invention may be at least one polypeptide of thefirst aspect. The formulation of the invention may be a formulation ofthe second aspect.

In a fourth aspect, the invention provides a polypeptide of theinvention or formulation of the invention for use in a method oftreating a disease. The polypeptide of the invention may be at least onepolypeptide of the first aspect. The formulation of the invention may bea formulation of the second aspect. The disease may be at least one ofType 1 and/or Type 2 diabetes, hyperglycemia, hypercholesterolemia,hypertension and metabolic syndrome. The disease may be Type 1 and/orType 2 diabetes. The disease may be hyperglycemia. The disease may behypercholesterolemia. The disease may be hypertension. The disease maybe metabolic syndrome.

In a fifth aspect, the invention provides a polypeptide of the inventionor formulation of the invention for use in a method of treating Type 1and/or Type 2 diabetes. The polypeptide of the invention may be at leastone polypeptide of the first aspect. The formulation of the inventionmay be a formulation of the second aspect. The use may provide areduction in hepatic glucose production, and/or a reduction incholesterol levels, and/or a reduction in glucagon levels, and/or areduction in blood pressure. The use may provide a reduction in hepaticglucose production. The use may provide a reduction in cholesterollevels. The use may provide a reduction in glucagon levels. The use mayprovide a reduction in blood pressure.

In a sixth aspect, the invention provides use of at least onepolypeptide according to the invention for the manufacture of amedicament for the treatment of Type 1 and/or Type 2 diabetes. Thepolypeptide according to the invention may be at least one polypeptideof the first aspect.

In a seventh aspect, the invention provides use of at least onepolypeptide according to the invention for the manufacture of amedicament for the treatment of at least one of hyperglycemia, and/orhypercholesterolemia, and/or hypertension, and/or metabolic syndrome.The polypeptide according to the invention may be at least onepolypeptide of the first aspect. The medicament may treat hyperglycemia.The medicament may treat hypercholesterolemia. The medicament may treathypertension. The medicament may treat metabolic syndrome.

In an eighth aspect, the invention provides a method for treating atleast one of diabetes, and/or hyperglycemia, and/orhypercholesterolemia, and/or hypertension in a subject, comprising:administering to the subject a polypeptide of the invention orformulation of the invention. The polypeptide of the invention may be atleast one polypeptide of the first aspect. The formulation of theinvention may be a formulation of the second aspect. The method maycomprise administering an effective amount of said polypeptide or saidformulation. The method may treat diabetes (e.g. Type 1 and/or Type 2diabetes). The method may treat hyperglycemia. The method may treathypercholesterolemia. The method may treat hypertension.

The present invention provides a pharmaceutical composition comprising atherapeutically effective amount of a RPS2 polypeptide or peptide analogcorresponding to one or more of SEQ ID NO(s) 1, 2, 3 or 4, and or moreof a pharmaceutically acceptable carrier, and/or one or more of apharmaceutically acceptable diluent, and/or one or more of apharmaceutically acceptable excipient. The polypeptide therapeutic ofthe present invention may be formulated for administration to a subjectin need of treatment as an oral formulation, a parenteral formulation, atopical formulation, an aqueous formulation, a solid formulation, alyophilized formulation, or a trans-dermal formulation.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph showing the relationship between body weight indiabetic animals (the Zucker diabetic rat animal model) treated withmetformin and/or IMG-1 administered intravenously (10 μg) and orally(200 μg, showing weight gain was consistent amongst the animals.

FIG. 2 is a graph showing that treatment of diabetic animals with IMG-1formulations (or and IV) resulted in normalized blood glucose levels,compared to control and metformin.

FIG. 3 is a graph showing that treatment of diabetic animals with IMG-1formulations (oral and IV) resulted in normalized blood pressure(systolic and diastolic) levels, compared to control and metformin.

FIG. 4 is a bar graph showing that treatment of diabetic animals withIMG-1 formulations (oral and IV) resulted in decreased HbA1c levels, ascompared to control and metformin.

FIG. 5 is a bar graph showing that treatment of diabetic animals withIMG-1 formulations (oral and IV) does not affect insulin levels(mcU/mL).

FIG. 6 is a bar graph showing that treatment of diabetic animals withIMG-1 formulations (oral and IV) resulted in decreased glucagon levels,compared to control and metformin.

FIG. 7 is a bar graph showing the mean cholesterol levels (mg/dL) asmeasured from diabetic animals administered IMG-1 (oral andintravenous), were lower as compared to untreated control. Treatedanimals averaged <170 mg/dL, whereas control measured 224 mg/dL.

FIG. 8 is a bar graph showing that IMG-1 formulations are effective atlowering blood glucose levels in a Type 1 diabetes animal model,reduction was significant as compared to control (untreated)diabetic-induced animals.

FIG. 9 is a graph showing the clearance of IMG-1 (measured as pg/mL)from serum over a 24 hour period in a Sprague Dawley animal model.Results shown are labeled as BES17-02-1, 2, 2 (indicated three animals).

FIG. 10 is a graph showing blood glucose levels in Sprague Dawleyanimals treated with IMG-1 formulations.

FIG. 11 is a graph showing that IMG-1 formulations administered at dosesranging from 1.0 μg to 1000.0 μg do not affect creatinine levels inSprague Dawley rats.

FIG. 12 is a graph showing that IMG-1 formulations administered at dosesranging from 1.0 μg to 1000.0 μg do not affect alanine aminotransferaselevels in Sprague Dawley rats.

FIG. 13 is a bar graph showing that IMG-1 formulations administered at adose of 2 μg (intravenous) decreases insulin resistance inDiabetic-Induced Obese (DIO) mice compared to control, based on glucoseinfusion rates (mg/kg/m).

FIG. 14 is a bar graph showing that IMG-1 formulations administered at adose of 2 μg (intravenous) inhibits hepatic glucose production (HGP) inDIO mice as compared to control.

FIG. 15 is a bar graph showing that IMG-1 formulations at a dose of 2 μg(intravenous) do not affect whole body glucose turnover, glycolysis orglycogen synthesis in DIO mice and are essentially equivalent tocontrol.

FIG. 16 is a bar graph showing that IMG-1 formulations at a dose of 2 μg(intravenous) do not affect skeletal muscle or white adipose glucoseuptake.

FIG. 17 is a bar graph showing the activity of truncated RPS2 peptides,designated as (IMG-1L and IMG-1S) compared to full-length RPS2 (IMG-1),and versus untreated. Formulations of IMG-1L, which correspond tofragments of the C-terminus of the full-length protein, exhibitedsimilar activity in vitro compared to IMG-1 (full length); whereas,formulations of IMG-1S (corresponding to the fragment of the N-terminusof the full-length protein) did not appear to exhibit activity, asmeasured by Optical Density from an MTT assay using endothelial cells.

DETAILED DESCRIPTION OF THE INVENTION

The following detailed description is provided to aid those skilled inthe art in practicing the present invention, but should not be construedto limit the present invention as modifications and variations in theembodiments disclosed herein may be made by those of ordinary skill inthe art without departing from the scope and spirit of the presentinvention. All publications and other references cited in thisapplication are hereby incorporated by reference in their entirety.

Definitions

The following terms are used in this disclosure to describe differentaspects of the invention. These terms are used for explanation purposesonly and are not intended to limit the scope for any aspect of theinvention.

As used herein “active ingredient”, “active compound”, “activecomponent”, and/or “active agent” may be used interchangeably and referto a polypeptide, peptide fragment, or analogue thereof having an aminoacid sequence comprising an amino acid sequence of SEQ ID NO. 1; or SEQID NO: 2, or SEQ ID NO: 3, SEQ ID NO: 4, or combinations thereof.Formulations of the present invention comprising the activeingredient/compound are collectively referred to as “IMG-1”formulations, without implying any specific dosage or concentration ofactive compound.

As used herein “effective amount” refers to that amount of the activeingredient/compound, which, when administered to a subject is effectiveto lower blood glucose levels to less than 200 mg/dl, lower cholesterolto less than 200 mg/dl, and/or lower blood pressure to less than 140/90mmHg.

As used herein “pharmaceutical formulation”, “pharmaceuticalcomposition”, “formulation”, or “composition” refer (interchangeably) toa liquid (aqueous, gel, or ointment), or a solid form containing anamount of active compound, which is prepared so that it is suitable foradministration to a mammal, such as a human or other animal, directly orafter reconstitution. If needed, the formulation may containpharmaceutically acceptable carriers and/or additives. For example,detergents/surfactants (e.g. PEG, Tween (20, 80, etc.), Pluronic),excipients, antioxidants (e.g. ascorbic acid, methionine), coloringagents, flavoring agents, preservatives, stabilizers, buffering agents,chelating agents (e.g. EDTA), suspending agents, isotonizing agents,binders, disintegrants, lubricants, fluidity promoters, and corrigents.The pharmaceutical compositions of the present invention may containother active ingredients in combination with the RPS2 polypeptide and/orpolypeptide analogues described herein.

As used herein, the terms “treat,” “treating” or “treatment,” and othergrammatical equivalents as used herein, include alleviating, abating orameliorating a disease or condition symptoms, preventing additionalsymptoms, ameliorating or preventing the underlying metabolic causes ofsymptoms, inhibiting the disease or condition, e.g., arresting thedevelopment of the disease or condition, relieving the disease orcondition, causing regression of the disease or condition, relieving acondition caused by the disease or condition, or stopping the symptomsof the disease or condition, and prophylaxis. The terms further includeachieving a therapeutic benefit and/or a prophylactic benefit. Bytherapeutic benefit is meant eradication or amelioration of theunderlying disorder being treated. Also, a therapeutic benefit isachieved with the eradication or amelioration of one or more of thephysiological symptoms associated with the underlying disorder such thatan improvement is observed in the patient, notwithstanding that thepatient may still be afflicted with the underlying disorder. Forprophylactic benefit, the compositions may be administered to a patientat risk of developing a particular disease, or to a patient reportingone or more of the physiological symptoms of a disease, even though adiagnosis of this disease may not have been made.

As used herein, a “therapeutically effective amount” to treat acondition, such as diabetes or hypertension, is an amount of activecompound capable of achieving a clinically-relevant end-point in apatient or patient population, such as reduced blood glucose levels indiabetes or reduced blood pressure in hypertension or reducedcholesterol in hypertriglyceridemia or hypercholesterolemia. Asnon-limiting examples, administration of an effective amount of an IMG-1composition has been shown in animal studies to lower blood glucose toless than 200 mg/dL (in a diabetes animal model); lower blood pressureto less than 140/90 mmHg; and lower total cholesterol to less than 200mg/dL.

The present invention provides a pharmaceutical composition for treatingone or more diseases in a subject, the diseases comprising one or moreof diabetes (Type I or Type 2), hypertension, hypercholesterolemia,vascular disease, or metabolic syndrome. The pharmaceutical compositionscomprise a purified or synthetic RPS2 polypeptide or peptide analoguecorresponding to one or more of SEQ ID NO 01, SEQ ID NO.: 2, SEQ ID NO:3, SEQ ID NO: 4, or active regions thereof, in combination with one ormore of a pharmaceutically acceptable carrier, diluent or excipient. Thepharmaceutical composition may be formulated for oral, parenteral ordermal/topical administration to a subject in need of treatment.

In another embodiment, the present invention provides an aqueouspharmaceutical formulation comprising a purified or synthetic RPS2peptide, analogue, or active region thereof, a buffer, such as phosphatebuffered saline (PBS), wherein the formulation has a pH within thenormal physiological range (approximately 7.4) and wherein the RPS2polypeptide or peptide analogue comprises an amino acid sequence setforth in one or more of SEQ ID NO 01, SEQ ID NO: 2, SEQ ID NO: 3, or SEQID NO: 4.

Also provided is a method of preventing, delaying the onset of, orreducing the severity of one or more conditions comprisinghyperglycemia, hypertension and/or hypercholesterolemia. The presentinvention also provides methods and compositions for improving proteinexpression in cell culture, particularly yeast, bacterial and mammaliancell culture, including media for growing cells for protein expressionand cell culture production media optimized for protein expression.

The present invention is also described through examples andexperimental results, which are intended as illustrative and notexhaustive, and shall be understood as not being limited thereto.

40 s RPS2 Peptide and Amino Acid Sequence(s)

Surprisingly, it has been discovered that a purified, isolated 40 sribosomal protein S2 (RPS2), including truncated fragments, exhibitstherapeutic benefits when administered to a subject, including: theability to reduce blood glucose levels; normalize blood glucose levels;lower blood cholesterol levels; reduce and normalize blood pressurelevels; decrease hemoglobin A1c levels; decrease glucagon levels; anddecrease insulin resistance. By way of reference, 40 s RPS2 is a proteinbelonging to the S5P family of ribosomal proteins. With respect tohumans, the RPS2 gene encodes a ribosomal protein that is a component ofthe 40S subunit and is located in the cytoplasm.

The RPS2 amino acid sequence does not contain a typical nuclearlocalization signal. Using deletion mutant analysis andrpS2-β-galactosidase chimeric proteins the putative identify of thenuclear targeting domains in RPS2 was determined. A central domaincomprising 72-75 amino acids is necessary and sufficient to target thechimeric β-galactosidase to the nucleus. The nuclear targeting domainshares no significant similarity to already-characterized nuclearlocalization signals in ribosomal proteins or other nuclear proteins.

The full length RPS2 amino acid sequence is a 293 amino acid sequencedesignated SEQ ID NO: 1; a truncated C-terminus fragment of SEQ ID NO: 1is a 159 amino acid fragment and is designated SEQ ID NO: 2; theunconventional nuclear localization signal is a 75 amino acid fragmentlocated between amino acids 161-235 of SEQ ID NO: 1, and is designatedSEQ ID NO: 3; an 87 amino acid fragment located between amino acids135-221 of SEQ ID NO: 1 is designated SEQ ID NO: 4; a truncatedN-terminus fragment of SEQ ID NO: 1 is a 134 amino acid fragment and isdesignated SEQ ID NO: 5.

RPS2 polypeptide of SEQ ID NO: 1 is 94%-100% homologous amongst allanimals when compared by sequence analysis performed utilizing the BLASTdatabase (https://blast.ncbi.nlm.nih.gov). The C-terminus regioncomprised of 159 amino acids of SEQ ID NO: 2 is 99-100% homologous forthe Animalia phylum. The 75 amino acid nuclear localization sequencecorresponding to SEQ ID NO: 3 shares 99-100% homology between allanimals and bacteria. Whereas animal/bacteria protein sequence is onaverage 98-100% homologous, homology to plants can be as low as 77%homologous.

An RPS2 amino acid sequence (293 amino acids) of the present inventionis shown below and designated SEQ ID NO: 01:

MADDAGAAGGPGGPGGPGMGNRGGFRGGFGSGIRGRGRGRGRGRGRGRGARGGKAEDKEWMPVTKLGRLVKDMKIKSLEEIYLFSLPIKESEIIDFFLGASLKDEVLKIMPVQKQTRAGQRTRFKAFVAIGDYNGHVGLGVKCSKEVATAIRGAIILAKLSIVPVRRGYWGNKIGKPHTVPCKVTGRCGSVLVRLIPAPRGTGIVSAPVPKKLLMMAGIDDCYTSARGCTATLGNFAKATFDAISKTYSYLTPDLWKETVFTKSPYQEFTDHLVKTHTRVSVQRTQAP AVATT

An RPS2 C-terminus fragment of the present invention is a 159 amino acidfragment of ≈18 kDa. The C-terminus fragment is shown below anddesignated SEQ ID NO: 2:

GHVGLGVKCSKEVATAIRGAIILAKLIVPVRRGYWGNKIGKPHTVPCKVTGRCGSVLVRLIPAPRGTGIVSAPVPKKLLMMAGIDDCYTSARGCTATLGNFAKATFDAISKTYSYLTPDLWKETVFTKSPYQEFTDHLVKTHTR VSVQRTQAPAVATT

A 75 amino acid nuclear localization signal of the present invention(corresponding to a sequence between amino acids 161-235 of SEQ IDNO: 1) is shown below and designated SEQ ID NO: 3:

SIVPVRRGYWGNKIGKPHTVPCKVTGRCGSVLVRLIPAPRGTGIVSAPVPKKLLMMAGIDDCYTSARGCTATLGN

An 87 amino acid sequence corresponding to a portion of the nuclearlocalization signal near the N-terminus between amino acids 135-221 ofSEQ ID NO: 1 is shown below and designated SEQ ID NO: 4

GHVGLGVKCSKEVATAIRGAIILAKLSIVPVRRGYWGNKIGKPHTVPCKVTGRCGSVLVRLIPAPRGTGIVSAPVPKKLLMMAGIDD

An RPS2 N-terminus fragment of the present invention is a fragment of≈13 kDa and designated SEQ ID NO: 5:

MADDAGAAGGPGGPGGPGMGNRGGFRGGFGSGIRGRGRGRGRGRGRGRGARGGKAEDKEWMPVTKLGRLVKDMKIKSLEEIYLFSLPIKESEIIDFFLGASLKDEVLKIMPVQKQTRAGQRTRFKAFVAIGDYN

Amino acids contained in the amino acid sequences in the presentinvention may be post-translationally modified according to methodsknown in the art. For example, the modification of an N-terminalglutamine (Gln) residue into a pyroglutamic acid (pGlu) residue bypyroglutamylation is well-known to those skilled in the art. Naturally,such post-translationally modified amino acids are included in the aminoacid sequences and within the scope of the present invention.

Purification of RPS2 and RPS2 Expression In Vitro

The RPS2 gene encoding the human 40S ribosomal protein S2, and variouspeptide portions, was subcloned into a pMAL-5 vector (New EnglandBiolabs). The RPS2 gene was amplified using a forward primer (sequence:atggcggatgacgccggtgc) and a reverse primer (sequence:ctatgttgtagccacagctgg) and the resulting PCR fragment was phosphorylatedand purified from low melt agarose. The resulting sequence was ligatedinto the pMAL-5 vector, and following ligation and incubation, spread onLB plates containing 100 μg/ml ampicillin and incubated overnight at 37°C. to generate transformant for in vivo protein production.

Generation of RPS2 Protein In Vivo

The RPS2 transformant was inoculated into 5 ml of broth and grown to2×108 cells/ml. This culture was used to inoculate a 200 mL LB amp 0.2%glucose to an A600 of around 0.5. The culture was then induced by addingITPG (isopropylthio-β-galactoside) to a final concentration of 0.3 mMand grown for an additional 4 h at 30° C. The cells were thencentrifuged and re-suspended in 25 ml column buffer per liter ofculture. The cells were lysed by freeze-thaw followed by passagingthrough a 20 gauge needle. The lysed cells were centrifuged and thesupernatant diluted by adding 125 ml cold CB for every 25 ml crudeextract. The diluted crude extract was added to a 15 ml amylose columnand washed with 12 column volumes of CB. The protein was eluted with CBand maltose (10 mM). The resulting elution was incubated with 1 μlFactor Xa diluted to 200 μg/ml for 4 hours at room temperature. Thefusion protein cleavage mixture was dialyzed at pH 8.0 and the amylasecolumns were washed with buffer and the fusion protein cleavage mixturewas loaded onto a column. The flow-through was collected and the amountof isolated protein was determined by bicinchoninic acid assay (BSA)assay and the amount of protein was assessed via ELISA.

For in vivo animal studies, a purified RPS2 protein corresponding to SEQID NO: 1 in solution following column purification was diluted with PBSto the desired concentrations (depending on oral or iv administration).The subsequent solution was then filtered through a 0.22 urn filter tofilter-sterilize the formulation for administration to animal subjects,as described in the Examples.

Similar studies were carried out with the gene for bacterial RPS2resulting in similar results in vivo as compared to human RPS2. Variousstudies were carried out to test the safety and efficacy of isolatedRPS2 polypeptide and peptide fragments as a therapeutic, includingtesting of IMG-1 formulations in a diabetic animal model, as well astoxicology studies in a control animal model. The studies performed andresults obtained are presented herein as Examples, along with accompanyFigures.

Example 1: Type 2 Diabetes Animal Model

The Zucker Diabetic Fatty (ZDF) strain of rat is widely known andcommonly used to study Type 2 Diabetes associated with obesity, as wellas hypertension and high cholesterol. The ZDF strain is an inbred ratmodel of early-onset diabetes in which all of the fa/fa male ratsdevelop diabetes at 10 to 12 weeks of age when fed a special diet ofPurina 5008 (Charles River Laboratories International, Inc., Wilmington,Mass., USA). The phenotype is homogeneous, mainly due to the fact thatthe strain is genetically inbred and that a special diet is provided.

Zucker Diabetic Fatty (ZDF) rats were fed a special diet of Purina 5008(Charles River Laboratories International, Inc.) to increase their bodyweight. Prior to the study the animals blood glucose levels wereassessed; only rats who had blood glucose levels greater than 200 mg/dlwere used in the study. The animals were randomly divided into 4 groups,untreated (n=7), once-daily metformin (200 mg/kg, n=7), 10 μg once-dailyIMG-1 intravenously (IV, n=8) and once-daily 200 μg IMG-1 orallyadministered (PO, n=9). The animals were maintained on this diet for 35days with their weight measured twice a week. After 35 days the animalswere sacrificed. FIG. 1 shows the distribution of body weight (grams)per group, measured over a thirty day period.

Fasting blood glucose (FBG) levels were assessed in the ZDF throughoutthe experiment using an Accutrend Plus System. ZDF rats treated withIMG-1 showed a marked decrease in blood glucose levels as early as 3days post treatment regardless of the modality administered, (averageFBG levels of 179 mg/dl for IV administered IMG-1 and 135 mg/dl POadministrated IMG-1 vs FBG levels of 281 mg/dl and 258 mg/dl in theuntreated and metformin treated groups respectively), with all IMG-1treated animals having normal FBG levels (levels below 200 mg/dl) by day7. Untreated controls and metformin treated animals had significantlyelevated FBG levels throughout the study with average levels exceeding400 mg/dl (481 mg/dl and 468 mg/dl respectively) at the end of thestudy. FIG. 2 shows that IMG-1 normalizes blood glucose levels in thediabetic animal model; blood glucose levels were lowered in animalstreated with IMG-1 versus control and metformin.

The blood pressure of the ZDF rats was monitored twice-weekly using atail cuff blood pressure monitor (CODA, Monitor from Kent Scientific).Within 3 days of treatment rats treated with IMG-1 started exhibitinglower blood pressure and ZDF rats who received IMG-1 orally had normalBP by day 7, while those who received intravenous IMG-1 had normal BP byday 10 (120/82 mmHg and 115/80 mmHg for IV and PO administered IMG-1respectively). Whereas the untreated and metformin treated ZDF rats hadhypertension (>140/90 mmHg, 141/95 mmHg untreated and 142/99 mmHgmetformin treated). FIG. 3 shows systolic and diastolic blood pressurelevels in animals treated with IMG-1 versus control and metformin;treatment with IMG-1 normalizes blood pressure in the diabetic animalmodel.

To assess HbA1c levels, blood draws were performed on all animals ondays 0, 15 and upon sacrifice on day 35. Bioassays were performed on theharvested blood and sera. Levels of Hemoglobin A1c (HbA1c) were measuredin all animals at all three time points. As shown in FIG. 4, the averageHb1Ac levels were not significantly different on day 0 (between 9.7 and10.8); at day 15 the levels of Hb1Ac were significantly lower in IMG-1treated groups compared to both untreated and metformin groups (10.2 IVand 9.5 PO vs 12.1 untreated and 11.0 metformin) and by day 35 IMG-1treated animals had significantly lower levels (7.4 IV and 7.5 PO) thanboth the untreated and metformin treated animals (12.3 untreated and11.8 metformin) as well as their own initial starting levels (10.4 IVand 10.5 PO).

Along with Hb1Ac levels, insulin levels were measured in all animals atthe three time points. As shown in FIG. 5, but unlike Hb1Ac levels shownin FIG. 4 there was no significant difference in insulin levels betweenuntreated and IMG-1 treated groups at any of the three time points.Moreover, the results seen in the control were consistent with theliterature.

Though there were no changes in the insulin levels of IMG-1, metforminand control animals, there was a significant decrease in the glucagonlevels of both IV and PO IMG-1 treated animals at days 15 (from 115μg/ml and 119 μg/ml to 90 μg/ml and 92 μg/ml respectively) and 35 (89μg/ml and 92 μg/ml); control and metformin treated animals had nosignificant difference in glucagon levels throughout the entire study.FIG. 6 shows IMG-1 decreases glucagon levels in the diabetic animalmodel; the decrease in glucagon levels as compared to control wasconsistent between oral and IV administered IMG-1.

The cholesterol levels of ZDF rats were assessed 48 hours following asingle dose injection of 20 μg IMG-1, after having continuous access toIMG-1 in the drinking water or untreated (n=4). IMG-1 treated animalshad significantly decreased levels of cholesterol compared to theuntreated cohort. FIG. 7 shows that IMG-1 decreases cholesterol in thediabetic animal model; untreated animals had a cholesterol level of 224mg/dL, while IMG-1 treated animals had cholesterol levels of 171 mg/dL(IV) and 156 mg/dL (oral), respectively.

Example 2: Type I Diabetes Animal Model

As IMG-1 was shown to normalize blood glucose levels, and decrease HA1Cin a Type 2 diabetic animal model, it was ascertained if IMG-1 couldaffect blood glucose levels in a Type/diabetic animal model. Diabetescan be induced in mice by using streptozotocin (STZ), a compound thathas a preferential toxicity toward pancreatic β cells and is a widelyused chemical for the induction of experimental diabetes in rodents. STZis an antibiotic produced by the bacterium Streptomyces achromogens andcontains a glucose molecule (in deoxy form) that is linked to a highlyreactive methylnitrosourea moiety that exerts a cytotoxic effects to thepancreatic β cells.

To investigate the efficacy of IMG-1 in a STZ-induced diabetes model 20male C57BL/6J mice at 3-4 months were administered STZ via IP injectionfor 5 days, in order to promote development of hyperglycemia. After STZinjection, pre-treatment (baseline) blood glucose levels were measuredafter a 4-hr fast, and used to select mice into 2 study groups: ControlGroup (placebo, n=7) and IMG-1 Group (n=8/group). The animals weretreated for 3 weeks with once daily oral gavage of Control or 33 μgIMG-1 PO. During the treatment phase the fasting blood glucose levelswas measured at 2-3 day intervals and the insulin and glucagon levelswere measured as well. At eighteen days post-STZ treatment it was shownthat fasting blood glucose levels of Control Group animals were elevatedfrom 216 mg/di to 319 mg/dl, and one of the Control Group animals (˜14%)had to be euthanized due to failure to thrive. However, the bloodglucose levels of the IMG-1 Group saw only a slight increase of glucoselevel, from 209 mg/dl to 237 mg/dl, and by day 18 it was shown that theIMG-1 Group had significantly lower blood glucose levels than thecontrol animals, as shown in FIG. 8.

Example 3: Toxicology and PK Studies

The Sprague Dawley model was utilized to access clearance and toxicologyof IMG-1 formulations. Three male Sprague Dawley rats were treated with20 μg active compound (corresponding to SEQ ID NO: 1) by intravenousadministration, followed by blood draws at intervals, specifically: 0, 5minutes (min), 15 min, 30 min, 60 min, 90 min, 2 hours (h), 4 h, 6 h, 24h and 48 h post-IMG-1 administration. Active compound was detected inthe blood of all three animals up to 2 h post administration, with athird animal displaying detectable levels up to 4 h post treatment, asshown in FIG. 9 by the serum level of IMG-1 active compound (pg/mL)measured over a 6 hour time period. Along with active compound level,blood glucose levels were assessed in the Sprague Dawley model. WhileIMG-1 administration dramatically decreased blood glucose levels in ZDFrats, a single dose of IMG-1 formulation (20 μg) administered to SpragueDawley rats did not promote hypoglycemia within 48 h (as shown in FIG.9) as seen by the blood glucose levels over a 48 hour period in theSprague Dawley animals.

To assess toxicity of IMG-1 formulations, a Pilot Dose-Finding ToxicityAssay was performed with Sprague Dawley rats. The testing consisted of 5groups, with 3 female and 3 male animals per group. Treated groups weregiven a single dose of one of the following IMG-1 concentrations: 1.0ug, 10 ug, 100 ug or 1000 μg. A control group received no treatment.Clinical observations were performed every 1 hour for 4 hours post-doseadministration, and daily for a total of 14 days for all 5 groups. Bloodwas drawn at 7 and 14 days post-administration for each of the 5 groups(treated and untreated). No adverse clinical observations were seen inany of the animals. Creatinine levels were measured to assess kidneyfunction and alanine aminotransferase levels were measured to assessliver function in each of the 5 groups. No discernable differencesbetween animals treated with IMG-1 formulation and untreated animals inboth creatinine and alanine aminotransferase levels were seen, as shownin FIGS. 10 and 11, respectively.

Example 4: Insulin Clamp Study

The most widely accepted research method for quantifying insulinsecretion and resistance is the euglycemic insulin clamp techniquemethod, which measures either how well an animal metabolizes glucose orhow sensitive an animal is to insulin. With this procedure, exogenousinsulin is infused, so as to maintain a constant plasma insulin levelabove fasting, while glucose is fixed at a basal level, (between 100-150mg/dl), by infusing glucose at varying rates.

To assess insulin action and glucose metabolism in IMG-1 treated animals(n=8) versus control (n=8), diet-Induced Obese (DIO) C57BL/6J miceunderwent a 2-h hyperinsulinemic-euglycemic clamping procedure. Prior toclamping, a 2 μg IMG-1 formulation was administered (per animal)intravenously via tail veins at 48 hours and 24 hours prior to the clamptest, compared to PBS treated animals. IMG-1-treated animals had asignificantly higher steady-state glucose infusion rates (38.8 mg/kg/mL)during clamping than PBS treated animals (30 mg/kg/mL). As shown in FIG.12, treatment with IMG-1 decreases insulin resistance in DIO mice.Hepatic Glucose Production (HGP) was also dramatically suppressed in allanimals treated with IMG-1, as shown by FIG. 13. Further, hepaticinsulin action (the percent suppression of HGP) was increased in allIMG-1-treated animals during the hyperinsulinemic-euglycemic clamp.However, IMG-1 did not affect whole-body glucose turnover, glycolysis orglycogen synthesis in any of the treated animals, as evidenced by FIG.14 showing the levels of glucose turnover, glycolysis and glycogensynthesis were nearly equal between the control animals and the treatedanimals. The results of the clamping procedure also showed that glucosemetabolism in skeletal muscle and adipose tissue were not significantlydifferent in either IMG-1 or PBS treated animals during the clamp assay,as evidenced by FIG. 15 which shows that skeletal muscle glucose uptakeand white adipose tissue glucose uptake were nearly identical betweenthe untreated and treated animals.

Example 5: Identification of RPS2 Active Regions

Full length purified RPS2 protein was digested with hydroxylamine(NH₂OH), which cleaved RPS2 at two positions: amino acid 134 at Asn inposition P1 and Gly in position P1′, resulting in two subunits,designated RPS2-short (IMG-1 s) of approximately 13 kDa, and RPS2-long(IMG-1L) of approximately 18 kDa. The resulting fragments were resolvedon a non-denaturing polyacrylamide gel, visualized and then electroeluted from the gel. The fragments were used to prepare formulations fortesting in cell culture.

Human Dermal Microvascular Endothelial Cells (CADMEC/HMVEC) provide anexcellent model system to study many aspects of endothelial function anddisease, especially those related to the microvasculature. The MU assayis a colorimetric assay for assessing cell metabolic activity.NAD(P)H-dependent cellular oxidoreductase enzymes may, under definedconditions, reflect the number of viable cells present. These enzymesare capable of reducing the tetrazolium dye MTT3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to itsinsoluble formazan, which has a purple color. In order to assess theeffect of IMG formulations on cellular activity, CADMEC cultures, suchas those available from Cell Applications, Inc., were treated with oneof the various formulations: 0.5 μg/mL full length protein (designatedIMG-1), 0.5 μg/mL of C-terminal fragment (designated IMG-1L), or 0.5μg/mL of N-terminal fragment (designated IMG-1 s). The treated cultureswere allowed to grow for 72 h. Following 72 h of cell growth, 100 μL ofcell cultures were treated with 10 μL of 12 mM MU, and incubated at 37°C. for 4 hours. Following the 4 h incubation with MTT, 100 μL of anSDS-HCl solution was added to the MU-treated cell cultures, in order tosolubilize the crystals, and was further incubated for an additional 4hrs. Absorbance was then read at 570 nm using a Microplate AbsorbanceSpectrophotometer (similar to the xMark™ from BioRad). As shown in FIG.17, at 72 h there was a significant increase in relative optical OD incells treated with formulations of IMG-1 and IMG-1L (164% and 157%respectively), while IMG-1S did not appear to affect cell growth anduntreated levels were in line with IMG-1S formulations. IMG-1Lcorrespond to fragments of the C-terminus of the full-length protein;whereas, formulations of IMG-1S correspond to the fragment of theN-terminus of the full-length protein. Thus, the C-terminus of RPS2 andfragments and/or analogues thereof are of therapeutic value, along withthe full length RPS2 protein.

Exemplary IMG-1 Formulations

The present invention provides pharmaceutical compositions comprisingone or more of a polypeptide, peptide, and/or analogue corresponding toone more of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:3 and/or SEQ ID NO:4.

Formulations of the present invention comprising a purified or syntheticpeptide or peptide analogue corresponding to SEQ ID: 01, SEQ 1 NO: 2,SEQ ID NO:3, and/or SEQ ID NO: 4 may be formulated according to methodsavailable to one of ordinary skill in the art. In one embodiment, apharmaceutical formulation comprises an RPS2 polypeptide or analogue, oran active peptide region thereof, corresponding to one of SEQ ID NO(s)1-4, in a range of less than 20 μg and up to 150 μg, as a solid doseform or a solution. In one embodiment, formulations comprise the peptidepresent at a concentration of 0.05 to 5 μg/L; in another embodimentformulations comprise peptide present at a concentration of 0.1 to 1μg/L; in yet another embodiment, formulations comprise peptide presentat a concentration of 50-150 μg/kg for an oral formulation and 5-15μg/kg for an intravenous formulation. Concentration of active ingredientand corresponding dosage will depend, in part, on the weight of thesubject, the route of administration, the symptoms/disorder to betreated, and the severity of the symptoms.

In one embodiment, a pharmaceutical formulation of the present inventionfurther comprises one or more absorption enhancers, including one ormore of detergents, surfactants, bile salts, Ca2+ chelating agents,fatty acids, medium chain glycerides, acyl carnitine, alkanoyl cholines,N-acetylated α-amino acids, N-acetylated non-α-amino acids, chitosans,mucoadhesive polymers, and phospholipids.

In one embodiment, exemplary excipients useful for the present inventioninclude: buffers, salts, surfactants,polyol/disaccharide/polysaccharides, amino acids, and antioxidants.Exemplary buffers that keep pH levels between 4.7 and 7.4 include:acetate, citrate, histidine, succinate, phosphate, andhydroxymethylaminomethane (Tris). Exemplary surfactants include:polysorbate 80 (Tween 80), polysorbate 20 (Tween 20), and poloxamer 188.Lyophilized (freeze-dried) formulations may use one or a mixture ofpolyol/disaccharide/polysaccharide (e.g., mannitol, sorbitol, sucrose,trehalose, and dextran 40). Sugars provide bulk for lyophilizedformulations and are known to serve as stabilizing agents fortherapeutic proteins. Sodium chloride (NaCl) is commonly used withprotein formulations. Exemplary antioxidants include: ascorbic acid,methionine, and ethylenediaminetetraacetic acid (EDTA).

In another embodiment, a pharmaceutical formulation of the presentinvention further comprises a surface modification by one or morelipophilic moieties. In yet another embodiment, a pharmaceuticalformulation comprises active agent optionally co-administered withconcentrated solution of one or more carrier molecules.

In yet another embodiment, a pharmaceutical formulation furthercomprises one or more synthetic bioadhesive polymers comprisingpolyacrylic acid or cellulose derivatives. Examples of polyacrylicacid-based polymers include, but are not limited to, carbopol,polycarbophil, polyacrylic acid (PAAc), polyacrylate, poly(methylvinylether-co-methacrylic acid), poly (2-hydroxyethylmethacrylate), poly(methacrylate), poly(alkylcyanoacrylate),poly(isohexylcyanoacrylate) and poly(isobutylcyanoacrylate). Cellulosederivatives include, but are not limited to, carboxymethyl cellulose,hydroxyethyl cellulose, hydroxypropyl cellulose, sodium carboxymethylcellulose, methylcellulose, and methylhydroxyethyl cellulose. Inaddition, seminatural bioadhesive polymers include chitosan and variousgums such as guar, xanthan, poly(vinylpyrrolidone), and poly(vinylalcohol).

In yet another embodiment, a pharmaceutical formulation of the presentinvention further comprises a gastrointestinal mucoadhesive patch system(GI-MAPS) comprising active agent with layered films contained in anenteric capsule, with a backing layer comprising a water-insolublepolymer, ethyl cellulose (EC); a surface layer comprising an entericpH-sensitive polymer such as hydroxypropylmethylcellulose phthalate,Eudragit L100 or S100; a coating layer comprising an adhesive layer; anda middle layer, peptide-containing layer, made of cellulose membraneattached to the backing layer. After oral administration, the surfacelayer dissolves at the targeted intestinal site and adheres to the smallintestinal wall, where a closed space is created on the target site ofthe gastrointestinal mucosa by adhering to the mucosal membrane. As aresult, both the active agent and the absorption enhancer coexist in theclosed space and a high-concentration gradient is formed between insidethe system and the enterocytes, which contributes to enhanced absorptionof peptide.

It will be clear to a person skilled in the art that features describedin relation to any of the embodiments described above can be applicableinterchangeably between the different embodiments. The embodimentsdescribed above are examples to illustrate various features of theinvention.

Throughout the description and claims of this specification, the words“comprise” and “contain” and variations of them mean “including but notlimited to”, and they are not intended to (and do not) exclude othermoieties, additives, components, or steps. Throughout the descriptionand claims of this specification, the singular encompasses the pluralunless the context otherwise requires. In particular, where theindefinite article is used, the specification is to be understood ascontemplating plurality as well as singularity, unless the contextrequires otherwise.

Features, characteristics, compounds, chemical moieties or groupsdescribed in conjunction with a particular aspect, embodiment or exampleof the invention are to be understood to be applicable to any otheraspect, embodiment or example described herein unless incompatibletherewith. All of the features disclosed in this specification(including any accompanying claims, abstract and drawings), and/or allof the steps of any method or process so disclosed, may be combined inany combination, except combinations where at least some of suchfeatures and/or steps are mutually exclusive. The invention is notrestricted to the details of any foregoing embodiments. The inventionextends to any novel one, or any novel combination, of the featuresdisclosed in this specification (including any accompanying claims,abstract and drawings), or to any novel one, or any novel combination,of the steps of any method or process so disclosed.

The reader's attention is directed to all papers and documents which arefiled concurrently with or previous to this specification in connectionwith this application and which are open to public inspection with thisspecification, and the contents of all such papers and documents areincorporated herein by reference.

1. An isolated polypeptide that comprises of a 40 s ribosomal protein S2(RPS2), or a fragment or analog thereof, comprising an amino acidsequence having at least 50% sequence identity to the amino acidsequence set forth in SEQ ID NO: 1, 2, 3 or
 4. 2. The polypeptide ofclaim 1, wherein the RPS2 or fragment or analog thereof has at least55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequenceidentity to the amino acid sequence set forth in SEQ ID NO: 1, 2, 3 or4.
 3. The polypeptide of claim 1, wherein the RPS2 or fragment or analogthereof is a synthetic polypeptide comprising the amino acid sequenceset forth in SEQ ID NO: 1, 2, 3 or
 4. 4. A pharmaceutical formulationcomprising at least one polypeptide or fragment or analog thereofaccording to claim
 1. 5. The pharmaceutical formulation of claim 4,wherein the formulation is an oral pharmaceutical formulation.
 6. Thepharmaceutical formulation of claim 4, wherein the formulation is apharmaceutical parenteral formulation.
 7. The pharmaceutical formulationof claim 4, wherein the formulation is a pharmaceutical topicalformulation.
 8. The pharmaceutical formulation of claim 4, wherein theformulation comprises one or more of a pharmaceutically acceptablecarrier, and/or one or more of a pharmaceutically acceptable diluent,and/or one or more of a pharmaceutically acceptable excipient.
 9. Thepharmaceutical formulation of claim 8, wherein the formulation is anaqueous pharmaceutical formulation.
 10. The pharmaceutical formulationof claim 8, wherein the formulation is a solid dose pharmaceuticalformulation.
 11. The pharmaceutical formulation of claim 8, wherein theaqueous pharmaceutical formulation comprises a buffer, optionally abuffer having a physiological pH.
 12. The pharmaceutical formulation ofclaim 11, wherein the buffer is phosphate buffered saline.
 13. Thepharmaceutical formulation of claim 4 for use in providing a reductionin hepatic glucose production, and/or a reduction in cholesterol levels,and/or a reduction in glucagon levels, and/or a reduction in bloodpressure, when administered to a subject in need thereof.
 14. Thepharmaceutical formulation of claim 8 for use in providing a reductionin hepatic glucose production, and/or a reduction in insulin resistance;and/or a reduction in cholesterol levels, and/or a reduction in glucagonlevels, and/or a reduction in blood pressure, when administered to asubject in need thereof.
 15. A method for treating at least one of Type1 and/or Type 2 diabetes, and/or hyperglycemia, and/orhypercholesterolemia, and/or hypertension, and/or metabolic syndrome ina subject, comprising: administering to the subject a compositioncomprising a therapeutically effective amount of a polypeptide, peptidefragment or analog thereof, according to claim
 1. 16. The pharmaceuticalformulation of claim 4, wherein the at least one polypeptide or fragmentor analog thereof consists of a polypeptide comprising the amino acidsequence set forth in SEQ ID NO: 2, 3 or 4, or mixtures thereof.
 17. Thepolypeptide, peptide fragment, or analog according to claim 1 for themanufacture of a pharmaceutical composition for the treatment of atleast one of hyperglycemia, and/or hypercholesterolemia, and/orhypertension, and/or metabolic syndrome.